Enzyme immunoassay for the quantitative determination of infliximab (Remicade ) in serum and plasma

Transcription

1 trace & catch Instructions for Use Infliximab (Remicade ) ELISA SHIKARI Q-INFLIXI Enzyme immunoassay for the quantitative determination of infliximab (Remicade ) in serum and plasma 0 REF TR-Q-INFLIXIv2 12 x 8 i 2-8 C Revision # 2.1 December 2016 Matriks Biotek Laboratories Distribuito in ITALIA da Li StarFish S.r.l. Via Cavour, Cernusco S/N (MI) telefono fax info@listarfish.it SHIKARI Q-INFLIXI 1

2 Contents Page Intended Use... 3 Summary and Explanation... 3 Test Principle... 4 Warnings and Precautions... 4 Storage and Stability of the Kit... 5 Specimen Collection and Storage... 5 Materials Supplied... 6 Materials Required but not Supplied... 6 Procedure Notes... 7 Pre-Test Setup Instruction... 8 Test Procedure... 9 Quality Control Calculation & Interpretation of Results Assay Characteristics Automation References Semi-Log Graph Paper Semi-Log Graph Paper SHIKARI Q-INFLIXI Free infliximab (Remicade ) quantitative analyses Required Volume (µl) 10 Total Time (min) 70 Sample Serum, plasma Sample Number 96 Detection Limit (ng/ml) 30 Spike Recovery (%) 97 Shelf Life (year) 1 2 SHIKARI Q-INFLIXI

3 Intended Use Enzyme immunoassay for the quantitative determination of free infliximab (Remicade ) in serum and plasma. Matriks Biotek Infliximab ELISA has been especially developed for the quantitative analysis of free infliximab in serum and plasma samples at high specificity. Summary and Explanation Infliximab (Remicade ) is a chimeric monoclonal antibody and used to treat autoimmune disorders. Infliximab reduces the amount of active human tumour necrosis factor alpha (htnfα) in the body by binding to it and preventing it from signaling the receptors for TNFα on the surface of various cell types. TNFα is one of the key cytokines that triggers and sustains the inflammatory reactions. Infliximab is used for the treatment of psoriasis, Crohn s disease, ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, ulcerative colitis, and approved by FDA. Single intravenous (IV) infusions of 3 mg/kg to 20 mg/kg showed a linear relationship between the dose administered and the maximum serum concentration. The volume of distribution at steady state was independent of dose and indicated that infliximab was distributed primarily within the vascular compartment. Median pharmacokinetic results for doses of 3 mg/kg to 10 mg/kg in rheumatoid arthritis and 5 mg/kg in Crohn s disease indicate that the terminal half-life of infliximab is 8.0 to 9.5 days. In controlled trials, clinical response rates of 20-40% have been achieved with abovementioned regimens in Crohn s disease and rheumatoid arthritis. However, the therapeutic benefits must be balanced against the risk of a variety of severe adverse events (e.g. severe infections including tuberculosis, hepatotoxicity, infusion reactions, serum sickness-like disease and lymphoma). The volume of distribution of infliximab is low (3-6 L) and represents the intravascular space. Elimination of infliximab is most probably accomplished through degradation by unspecific proteases. It seemed that methotrexate delayed the decline in the serum concentrations of infliximab. When relating serum concentrations to the clinical response in patients, it can be assumed that through concentrations above 1mg/mL could be used as a kind of therapeutic target. The rate of clinical remission was higher for patients with a detectable trough serum infliximab compared with patients in whom serum infliximab was undetectable, including those without antibodies. A detectable trough serum infliximab was also associated with a lower C-reactive protein and a higher rate of endoscopic improvement. For Crohn s disease patients treated with scheduled maintenance SHIKARI Q-INFLIXI 3

4 infusions of infliximab, the serum concentration of infliximab seemed to predict clinical outcome. It was also proposed that, the surveillance of circulating infliximab concentration during maintenance therapy represents an indirect but reliable method to monitor anti-infliximab immunization. In this context, identification of biomarkers for (non-)response and risk factors for adverse drug reactions relating to serum concentrations and therapeutic drug monitoring of infliximab would be very helpful. Test Principle Solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle. Standards and samples (serum or plasma) are incubated in the microtitre plate coated with the reactant for infliximab (Remicade ). After incubation, the wells are washed. A horse radish peroxidase (HRP) conjugated probe is added and binds to infliximab captured by the reactant on the surface of the wells. Following incubation wells are washed and the bound enzymatic activity is detected by addition of chromogen-substrate. The colour developed is proportional to the amount of infliximab in the sample or standard. Results of samples can be determined directly using the standard curve. Warnings and Precautions 1. For for professional use only and professional use only. 2. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood. For further information (clinical background, test performance, automation protocols, alternative applications, literature, etc.) please refer to the local distributor. 3. In case of severe damage of the kit package please contact Matriks Biotek or your supplier in written form, latest one week after receiving the kit. Do not use damaged components in test runs, but keep safe for complaint related issues. 4. Obey lot number and expiry date. Do not mix reagents of different lots. Do not use expired reagents. 5. Follow good laboratory practice and safety guidelines. Wear lab coats, disposable latex gloves and protective glasses where necessary. 4 SHIKARI Q-INFLIXI

5 6. Reagents of this kit containing hazardous material may cause eye and skin irritations. See MATERIALS SUPPLIED and labels for details. 7. Chemicals and prepared or used reagents have to be treated as hazardous waste according the national biohazard safety guidelines or regulations. 8. Avoid contact with Stop solution. It may cause skin irritations and burns. 9. All reagents of this kit containing human serum or plasma (i.e. standards) have been tested and were found negative for HIV I/II, HBsAg and HCV. However, a presence of these or other infectious agents cannot be excluded absolutely and therefore reagents should be treated as potential biohazards in use and for disposal. 10. Some reagents contain sodium azide (NaN 3 ) as preservatives. In case of contact with eyes or skin, flush immediately with water. NaN 3 may react with lead and copper plumbing to form explosive metal azides. When disposing reagents, flush with large volume of water to avoid azide build-up. Storage and Stability The kit is shipped at ambient temperature and should be stored at 2-8 C. Keep away from heat or direct sun light. The strips of microtiter plate is stable up to the expiry date of the kit in the broken, but tightly closed bag when stored at 2 8 C. Specimen Collection and Storage Serum, Plasma (EDTA, Heparin)* The usual precautions for venipuncture should be observed. It is important to preserve the chemical integrity of a blood specimen from the moment it is collected until it is assayed. Do not use grossly hemolytic, icteric or grossly lipemic specimens. Samples appearing turbid should be centrifuged before testing to remove any particulate material. Storage: 2-8 C -20 C Stability: 2 d 6 mon Keep away from heat or direct sun light Avoid repeated freeze-thaw cycles *. Infliximab (Remicade ) infusion camouflages/masks the presence of antibody to infliximab (ATI) in serum/plasma samples. Therefore, blood sampling time is critical for detection of ATI. The Matriks Biotek Laboratories suggests to obtain blood sample just before the infusion of infliximab (Remicade ) or at least 2 weeks after the infusion of infliximab (Remicade ). SHIKARI Q-INFLIXI 5

6 Materials Supplied 1 x 12 x 8 MTP 5 x 0.3 ml STND A-E 1 x 50 ml ASSAY BUF 1 x 12 ml HRP CONJ 1 x 12 ml TMB SUBS 1 x 12 ml TMB STOP 1 x 50 ml 2 x 1 FOIL WASHBUF CONC Microtiter Plate Break apart strips. Microtiter plate with 12 rows each of 8 wells coated with reactant. Infliximab Standards A-E 3; 1; 0.3; 0.1; 0 microgram/ml Ready to use. Used for construction of the standard curve. Contains infliximab (Remicade ), human serum, stabilizer and <0.1% NaN 3. Assay Buffer Blue colored. Ready to use. Contains proteins and <0.1% NaN 3. Horse radish peroxidase-conjugated Probe Red colored. Ready to use. Contains HRP-probe, stabilizer and <0.1% NaN 3. TMB Substrate Solution Ready to use. Contains TMB TMB Stop Solution Ready to use. 1N HCl. Wash Buffer, Concentrate (20x) Contains Buffer with Tween 20. Adhesive Foil For covering of Microtiter Plate during incubation. 2x 1 SLGP Semi-Log Graph Paper For constructing standard curve and calculation of results. Materials Required but not Supplied 1. Micropipettes (Multipette Eppendorf or similar devices, < 3% CV). 2. Calibrated measures. 3. Tubes (1 ml) for sample dilution. 4. Wash bottle, automated or semi-automated microtiter plate washing system 5. Microtiter plate reader capable of reading absorbance at 450/650 nm. 6. Bidistilled or deionised water, paper towels, pipette tips and timer. 6 SHIKARI Q-INFLIXI

7 Procedure Notes 1. Any improper handling of samples or modification of the test procedure may influence the results. The indicated pipetting volumes, incubation times, temperatures and pretreatment steps have to be performed strictly according to the instructions. Use calibrated pipettes and devices only. 2. Once the test has been started, all steps should be completed without interruption. Make sure that required reagents, materials and devices are prepared ready at the appropriate time. Allow all reagents and specimens to reach room temperature (18-25 C) and gently swirl each vial of liquid reagent and sample before use. Mix reagents without foaming. 3. Avoid contamination of reagents, pipettes and wells/tubes. Use new disposable plastic pipette tips for each reagent, standard or specimen. Do not interchange caps. Always cap not used vials. Do not reuse wells/tubes or reagents. 4. Use a pipetting scheme to verify an appropriate plate layout. 5. Incubation time affects results. All wells should be handled in the same order and time sequences. It is recommended to use an 8-channel Micropipettor for pipetting of solutions in all wells. 6. Microplate washing is important. Improperly washed wells will give erroneous results. It is recommended to use a multichannel pipette or an automatic microplate washing system. Do not allow the wells to dry between incubations. Do not scratch coated wells during rinsing and aspiration. Rinse and fill all reagents with care. While rinsing, check that all wells are filled precisely with Wash Buffer, and that there are no residues in the wells. 7. Humidity affects the coated wells/tubes. Do not open the pouch until it reaches room temperature. Unused wells/tubes should be returned immediately to the resealed pouch including the desiccant. SHIKARI Q-INFLIXI 7

8 Pre-Test Setup Instructions 1. Preparation of Components Dilute/ disolve Component with Diluent Relation Remarks Storage Stability 10 ml Wash Buffer* Up to 200 ml bidist. Water 1:20 Warm up at 37 C to dissolve crystals. Mix vigorously. *. Prepare Wash Buffer before starting assay procedure. 2. Dilution of Samples* 2-8 C 4 w Sample To be diluted With Relation Remarks Serum/ Plasma Initially 1:20 Assay Buffer 1:20-1:100 For dilution at 1:20; 10µL Sample + 190µL Assay Buffer For dilution at 1:100; 5µLSample + 495µL Assay Buffer *. Patient samples with a concentration of infliximab above the measuring range are to be rated as > highest standard. The result must not be extrapolated. The patient sample in question should be further diluted with Assay Buffer and retested. 8 SHIKARI Q-INFLIXI

9 Test Procedure 1 Pipette 100µl of Assay Buffer non-exceptionally into each of the wells to be used. Pipette 10 µl of each ready-to use Standards, and Diluted Samples into the respective wells of microtiter plate Wells A1: Standard A B1: Standard B C1: Standard C D1: Standard D E1: Standard E F1 and on: Sample (Serum/Plasma) Cover the plate with adhesive foil. Incubate 30 min at room temperature (18-25 C). Remove adhesive foil. Discard incubation solution. Wash plate 3 times each with 300 µl of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel. 5 Pipette 100 µl of ready-to use HRP-Conjugated Probe into each well. 6 7 Cover the plate with adhesive foil. Incubate 30 min at room temperature (18-25 C). Remove adhesive foil. Discard incubation solution. Wash plate 3 times each with 300 µl of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel. 8 Pipette 100 µl of TMB Substrate Solution into each well Incubate 10 min (without adhesive foil.) at room temperature (18-25 C) in the dark. Stop the substrate reaction by adding 100 µl of Stop Solution into each well. Briefly mix contents by gently shaking the plate. Color changes from blue to yellow. Measure optical density with a photometer at 450/650 nm within 30 min after pipetting of the Stop Solution. SHIKARI Q-INFLIXI 9

10 Quality Control The test results are only valid only if the test has been performed following the instructions. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable standards/laws. All standards must be found within the acceptable ranges as stated above and/or label. If the criteria are not met, the run is not valid and should be repeated. In case of any deviation the following technical issues should be proven: Expiration dates of (prepared) reagents, storage conditions, pipettes, devices, incubation conditions and washing methods. Calculation & Interpretation of Results 1. A standard curve should be calculated using the standard concentration (X-axis) versus the OD450 (we suggest that OD readings done at double wavelength at OD450/650 and OD value read at 650nm subtracted from 450nm OD VALUE) VALUES (Y-axis).In case of manual plot, we suggest semilog graph paper. If computer data regation is going to be used, we recommend primarily "4 Parameter Logistic (4PL)" or secondly the "point-to-point calculation". 2. The concentration of the samples can be read from this standard curve as follows. Using the absorbance value for each sample, determine the corresponding concentration of the drug from standard curve. This value always has to be multiplied by the dilution factor. If any diluted sample is reading greater than highest standard, it should be further diluted appropriately with Assay Buffer and retested. Also this second dilution has to be used for calculation the final results. 3. Any sample diluted at 1:20 and still reading greater than the highest standard should be further diluted appropriately with Assay Buffer and retested. Because the samples have been diluted, the concentration determined from the standard- curve must be multiplied by the dilution factor. 10 SHIKARI Q-INFLIXI

11 Typical Calibration Curve (Example. Do not use for calculation!) Standard Concentration (µg/ml) Mean OD450/650 A 3 2,627 B 1 1,390 C 0,3 0,487 D 0.1 0,177 E 0 0,044 SHIKARI Q-INFLIXI 11

12 Assay Characteristics 1. Specificity: Except for the other therapeutic anti-tnf antibodies such as etanercept (Enbrel ) and/or adalimumab (Humira ) with which cross reaction might occur to some extends, there is no cross reaction with native serum immunoglobulin. 2. Sensitivity: The lowest detectable level that can be distinguished from the zero standard is 30 ng/ml. 3. Precision Of Kit: Intra-assay CV: <20% for infliximab range mg/ml. Inter-assay CV: <20% for infliximab range mg/ml. 4. Recovery: Recovery rate was found to be equal and higher than 98% with normal human serum samples with known concentrations. Automation Experiments have shown that the infliximab ELISA is also suitable to run on an automated ELISA processor. 12 SHIKARI Q-INFLIXI

13 References 1. Elliott MJ, Maini RN, Feldmann M, Long-Fox A, Charles P, Bijl H, Woody JN, Repeated therapy with monoclonal antibody to tumour necrosis factor alpha (ca2) in patients with rheumatoid arthritis, Lancet, 1994; Oct 22;344(8930): Elliott MJ, Maini RN, Feldmann M, Kalden JR, Antoni C, Smolen JS, Leeb B, Breedveld FC, Macfarlane JD, Bijl H, et al., Randomised double-blind comparison of chimeric monoclonal antibody to tumour necrosis factor alpha (ca2) versus placebo in rheumatoid arthritis. Lancet, Oct 22;344(8930): Keating GM, Perry CM, infliximab: an updated review of its use in Crohn s disease and rheumatoid arthritis, BioDrugs, 2002;16(2): Lyseng-Williamson KA, Foster RH, infliximab: a pharmacoeconomic review of its use in rheumatoid arthritis, Pharmacoeconomics, 2004;22(2): Maini RN, Elliott MJ, Brennan FM, Williams RO, Chu CQ, Paleolog E, Charles PJ, Taylor PC, Feldmann M, Monoclonal anti-tnf alpha antibody as a probe of pathogenesis and therapy of rheumatoid disease, Immunol Rev, 1995 Apr;144: Xu Z, Seitz K, Fasanmade A, Ford J, Williamson P, Xu W, Davis HM, Zhou H, Population pharmacokinetics of infliximab in patients with ankylosing spondylitis, J Clin Pharmacol, 2008 Jun;48(6): Epub 2008 Apr Caviglia R, Boskoski I, Cicala M, Long-term treatment with infliximab in inflammatory bowel disease: safety and tolerability issues, Expert Opin Drug Saf, 2008 Sep;7(5): Reddy JG, Loftus EV Jr, Safety of infliximab and other biologic agents in the inflammatory bowel diseases, Gastroenterol Clin North Am, 2006 Dec;35(4): Klotz U, Teml A, Schwab M, Clinical pharmacokinetics and use of infliximab, Clin Pharmacokinet, 2007; 46(8): Maser EA, Villela R, Silverberg MS, Greenberg GR, Association of trough serum infliximab to clinical outcome after scheduled maintenance treatment for Crohn s disease, Clin Gastroenterol Hepatol, 2006 Oct;4(10): Reimold AM, New indications for treatment of chronic inflammation by TNF-alpha blockade, Am J Med Sci, 2003 Feb;325(2): Ternant D, Mulleman D, Degenne D, Willot S, Guillaumin JM, Watier H, Goupille P, Paintaud G, An enzyme-linked immunosorbent assay for therapeutic drug monitoring of infliximab, Ther Drug Monit, 2006 Apr;28(2): Candon S, Mosca A, Ruemmele F, Goulet O, Chatenoud L, Jean-Pierre Ce zard, Clinical and biological consequences of immunization to infliximab in pediatric Crohn s disease, Clinical Immunology, 2006; 118: Gottlieb AB, Masud S, Ramamurthi R, Abdulghani A, Romano P. Et al., Pharmacodynamic and pharmacokinetic response to anti-tumor necrosis factor-monoclonal antibody (infliximab) treatment of moderate to severe psoriasis vulgaris, J Am Acad Dermatol 2003;48: SHIKARI Q-INFLIXI 13

14 15. Maini RN, Breedveld FC, Kalden JR, Smolen JS, et al., Therapeutic efficacy of multiple intravenous infusions of anti-tumor necrosis factor a monoclonal antibody combined with low-dose weekly methotrexate in rheumatoid arthritis, Arthritis & Rheumatism, 1998; 41: Xu Z, Seitz K, Fasanmade A, Ford J, Williamson P, Xu W, Davis HM, et al. Population Pharmacokinetics of infliximab in Patients With Ankylosing Spondylitis, J. Clin. Pharmacol. 2008; 48; SHIKARI Q-INFLIXI

15 OD 450/650 nm SHIKARI Q-INFLIXI 15

16 OD 450/650 nm 16 SHIKARI Q-INFLIXI